Manufacture of microspheres using a hydrocyclone

ABSTRACT

This disclosure features a system for processing microspheres. A vessel contains a suspension of solidified microspheres comprising polymer and an active agent. A hydrocyclone has a fluid inlet, a first fluid outlet and a second fluid outlet. The fluid inlet is in fluid communication with the vessel and receives the suspension. The second fluid outlet contains a flow of the suspension having concentrated microspheres. The first fluid outlet contains a flow of a relatively large amount of liquid compared to the flow from the second fluid outlet. Also featured is a method of processing the microspheres using the hydrocyclone.

TECHNICAL FIELD

The field is the manufacture of sustained release pharmaceuticalproducts.

TECHNICAL BACKGROUND

Sustained release injectable microparticles have attracted attention dueto several advantages. They have high patient compliance and conveniencedue to less frequent injections. Higher efficacy was generally achievedwith lower dose due to the maintenance of sustained and effectiveconcentration of the drug in the blood. If necessary, these formulationscould be used to achieve higher local concentration to treat specificdiseases. However, manufacturing these dosage forms is challenging sincethe product should be sterile. Standard sterilization methods do notwork with these products or adversely affect the quality of the product.Hence the product should be manufactured under aseptic conditions.Aseptic manufacturing of a microparticulate product is challenging. Itwas established that microparticles could be manufactured by acontinuous process as reported in several patents (U.S. Pat. Nos.5,945,126; 6,270,802; 6,361,798; 7,300,671 and 6,939,033). When makingmicrospheres, it is preferred to produce the particles in a continuousprocess, such as concentrating the particles to the desired level,removing undesired particles and also removing undesired solvents andsurfactants by washing. To produce microparticles in a continuous flowprocess, equipment is available at various size ranges. For example,in-line dynamic mixers are available at various sizes from manufacturerssuch as Silverson machines and Ross mixers. Similarly, in-line staticmixers are available from companies such as Ross mixers, Sulzer andKomax at various sizes. However, there are not many equipment options toprocess the microspheres at such a faster rate. Processing involvesremoving the unwanted components such as excess continuous phase thatalso contains solvents and non-encapsulated drugs. Equipment such asdead-end filtration (e.g., PharmSep from Swecco, Stir-Cell assembly fromMillipore), continuous flow centrifuges (centrifuges from Alfa Lavel)and trans-membrane filtration (e.g., hollow fiber filters from GEHealthcare, Spectrum) can process microsphere suspensions. However,dead-end filtration cannot handle large volumes, requires a huge surfacearea and uses cumbersome equipment with moving parts. Also, clogging offilters and sieves is a common problem during dead end filtrationsacrificing the efficiency during the process. For larger scaleoperations it is necessary to change the clogged filter/sieve and thisintervention could affect the aseptic operation. Continuous flowcentrifuges may have a problem of particle packing (aggregation) andalso are difficult to operate under aseptic conditions. Trans-membranefiltration has a limitation due to the huge surface area requirementsand very high flow rate requirement for re-circulation. Filter membraneclogging is also a problem for large scale operations. Additionally,removal of small particles from the product becomes necessary in severalsituations since smaller particles could cause inflammation due tomacrophages. Spin filters from Sartorius and vibrating sieves fromSwecco are the available options to remove small particles. However,sieve clogging is the major problem for both devices; therefore they arenot widely used.

TECHNICAL SUMMARY

A hydrocyclone is used because it is capable of processing large volumesrapidly and can be adapted for aseptic manufacturing. The hydrocycloneoccupies minimum space in the aseptic area, does not have any movingparts and can process the microsphere suspension at a faster rateeliminating deficiencies with existing equipment. Additionally, it wasfound that the hydrocyclone can remove an unwanted fraction of particlesthat can compromise the quality of the product. While the termmicrospheres is used throughout this disclosure it is understood thatthis term is interchangeable with the terms microparticles andmicrocapsules since the particles may not be strictly spherical and theactive agent can be dispersed throughout or encapsulated by the polymer.

Manufacturing of microspheres produces particles with a wide particlesize distribution. Smaller particles can pose problems such as initialburst and faster release rate. Small particles may not contain higherdrug load compared to larger particles. Additionally, smaller particlesmay provoke an immune response due to macrophage attack. Therefore, forcertain applications it is important that the formulation is free fromparticles, for example, less than 10 microns, preferably less than 5microns. As one example, for applications of microparticles to treatosteorarthritis by injecting in synovial tissue, the product should befree from particles less than 20 microns. The hydrocyclone can removethese small particles.

Even though removal of unwanted particle sizes can also be achieved bysieving processes, sieving is difficult under aseptic manufacturingconditions, especially removing a fraction of particles from theproduct. The hydrocyclone can be used to process the microspheresproduced at a faster rate by eliminating the continuous phase at a muchfaster rate and eliminating smaller particles. This device can also beused to eliminate larger particles. The hydrocyclone can be combinedwith transmembrane filtration (e.g., a hollow fiber filter) so that thehollow fiber filter can be operated efficiently. Since most of thecontinuous phase is eliminated by the hydrocyclone it is easy to operatethe hollow fiber filter at a lower flow rate (recirculation rate) withsimple and smaller pumps such as peristaltic pumps. Additionally,smaller particles that could clog the filter membrane by blocking thepores can be eliminated by the hydrocyclone. By removing a huge fractionof these smaller particles using a hydrocyclone, the operating life ofthe filter membrane can be extended, thus enhancing the ability toproduce large batch sizes. This is useful during aseptic manufacturingsince replacing the hollow fiber filter can compromise the asepsis ofthe process which could lead to rejecting the entire batch.

Similarly, by eliminating a large fraction of smaller particles from themicrosphere suspension by hydrocyclone operation the microspheresproduced could be processed with less difficulty using dead endfiltration (e.g., Swecco sieves). For information about dead endfiltration see: AAPS Pharm Sci Tech, 2001, 2(1); US20090104274. If thesuspension is substantially free from small particles the processing(concentrating, washing, achieving final formulation) can be performedusing the sieve system also.

Microspheres are produced by combining the dispersed phase andcontinuous phase under the influence of mixing. By selecting theappropriate ratio of the dispersed phase to the continuous phase theoutput can be a suspension of solidified microspheres or an emulsion. Ifit is an emulsion, additional fluid can be added to solidify theemulsion by solvent extraction. This could be performed continuously oras a batch process. During the continuous microparticle manufacturingprocess the suspension output rate can be 1 to 50 L/min depending uponthe in-line mixing system. Particle concentration in the suspension canbe 0.1 g/L to 100 g/L. Elimination of continuous phase and concentratingmicroparticles in the suspension are necessary steps during thepharmaceutical preparation. This can be performed by several proceduresand associated equipment such as dead-end filtration, centrifugation andtrans-membrane filtration, which when used alone have limitations asexplained.

In order to increase the rate of microsphere processing whilemaintaining favorable properties of the microspheres and aseptic natureof the product, the hydrocyclone was found useful. The hydrocyclonesignificantly increased the process capability and the quality of themicrospheres. The hydrocyclone can be easily implemented and used atseveral different points within the overall process. The hydrocyclonecan be used to remove the excess continuous phase (CP) with or withoutthe removal of small particles, to remove larger particles and alsoduring the washing step to remove CP and unwanted components such asresidual solvents and non-encapsulated drug.

The hydrocyclone can have an important effect on a sustained releaseformulation to improve not only its concentration for processingefficiency, but also to help provide formulations which are safer andmore effective. The hydrocyclone may be able to help select a preferredparticle size distribution in the product. This selection can reduce afraction that is potentially harmful (or undesirable). Specifically,smaller particles can be reduced or eliminated. These smaller particlescan be a potential source of a macrophage attack at the site ofinjection. The hydrocyclone is capable of processing the microspheresuspension at a much faster rate removing unwanted components comparedto other devices.

For fast processing, more than one hydrocyclone can be used in parallel.For good yield more than one hydrocyclone can be used in series. Thereare several variables in the hydrocyclone that could change theprocessing efficiency and yield. For a given hydrocyclone differentfixed angle apex identifications (IDs) can be selected to manipulate thebottom flow and overflow.

Turning now to a discussion of specific aspects of this disclosure, afirst embodiment pertains to a system for processing microspherescomprising a vessel and a hydrocyclone. The vessel contains a suspensionof solidified microspheres comprising polymer and active agent. Thehydrocyclone has a fluid inlet, a first fluid outlet and a second fluidoutlet. The fluid inlet is in fluid communication with the vessel andreceives the suspension. The second fluid outlet contains a flow of thesuspension having concentrated microspheres. The first fluid outletcontains a flow of a relatively large amount of liquid compared to theflow from the second fluid outlet.

Referring to particular features of the first embodiment, the system cancomprise a pump disposed between the vessel and the hydrocylone forpumping the suspension from the vessel to the fluid inlet underpressure. The system can comprise a mixer that combines the polymer,solvent and the active agent to form an emulsion. The mixer is in fluidcommunication with the vessel and the emulsion is solidified in thevessel to form the suspension in the vessel. Alternatively, the systemcan comprise another mixer that combines the polymer, solvent and theactive agent to form the suspension. The mixer is in fluid communicationwith the vessel.

A second embodiment of this disclosure features a system forconcentrating microspheres comprised of polymer and active agent. Thesystem comprises a mixer and a hydrocyclone. A microsphere suspensioncould be produced by mixing in the mixer a dispersed phase and acontinuous phase. The dispersed phase includes solvent, polymer and anactive agent. The mixer includes a mixing element which mixes thedispersed phase and the continuous phase to make a suspension ofmicrospheres. The hydrocyclone has a fluid inlet, a first fluid outletand a second fluid outlet. The fluid inlet is in fluid communicationwith the mixer and receives the suspension. The second fluid outletcontains a flow of the suspension having concentrated microspheres andthe first fluid outlet contains a flow of a relatively large amount ofliquid compared to the flow from the second fluid outlet. Even thoughthe microspheres produced by the mixer could be directly processed bythe hydrocyclone, for control purpose it could be performed in two stepswith an intermediate vessel receiving the suspension from the mixer andfrom which a delivery pump will deliver the suspension to thehydrocyclone.

Now, more specific aspects of the second embodiment will be addressed.The system comprises: a vessel for containing and stirring thesuspension; first tubing leading from the mixer to the vessel; a sourceof water or a suspending medium; second tubing leading from the sourceto the vessel; a pump for moving water or suspending medium from thesource along the second tubing into the vessel; third tubing leadingfrom the vessel to the fluid inlet; and a pump for pumping thesuspension from the vessel to the fluid inlet

The flow from the first fluid outlet can contain a relatively largeamount of continuous phase, water or suspending medium as the liquid,compared to the flow from the second fluid outlet.

The flow from the first fluid outlet can contain a relatively largeamount of fine microspheres compared to the flow from the second fluidoutlet.

The system can also comprise a second hydrocyclone (HC-2) having a fluidinlet, a first fluid outlet and a second fluid outlet. The HC-2 secondfluid outlet contains a flow of the suspension having concentratedmicrospheres and the HC-2 first fluid outlet contains a flow of arelatively large amount of the liquid compared to the flow from the HC-2second fluid outlet. The first fluid outlet of the hydrocyclone (HC-1)is in fluid communication with the fluid inlet of the secondhydrocyclone (HC-2) and the concentrated suspension from the HC-1 secondfluid outlet and from the HC-2 second fluid outlet are combined

The flow from the first fluid outlet of the second hydrocyclone (HC-2)can contain a relatively large amount of the continuous phase, the wateror the suspending medium compared to the flow from the HC-2 second fluidoutlet.

The system can comprise a second hydrocyclone in series or parallel withthe hydrocyclone.

The system can further comprise a solvent removal vessel (SRV) thatreceives the combined concentrated suspension from the HC-1 second fluidoutlet and from the HC-2 second fluid outlet to achieve washedmicrospheres with lower residual solvent and free from unwantedcomponents such as surfactant in the CP and non-encapsulated drug.

The system can comprise: a hollow fiber filter (HFF) having a HFF inlet,a first HFF outlet and a second HFF outlet; fourth tubing between thesolvent removal vessel (SRV) and the HFF inlet; and a pump for movingthe solvent-removed suspension from the SRV, along the fourth tubing tothe HFF inlet and fifth tubing extending from the second HFF outlet tothe SRV. Permeate is removed from the first HFF outlet, and filteredsuspension travels from the second HFF outlet along the fifth tubing tothe SRV.

The system can comprise a second source of water or suspending medium,sixth tubing leading from the second source to the solvent removalvessel (SRV) and a pump for pumping the water or the suspending mediumfrom the second source along the sixth tubing into the SRV.

The system can comprise a hollow fiber filter that receives themicrosphere suspension traveling from the second fluid outlet of one ormore hydrocyclones.

The system can comprise: a wet sieve having a suspension inlet, a liquidoutlet and a microsphere outlet; a source of water or suspending medium;tubing leading from the source to the sieve; and a pump for moving thewater or the suspending medium from the source along the tubing into thesieve. The suspension inlet of the sieve receives the concentratedsuspension from the second fluid outlet of one or more hydrocyclones.

A third embodiment of this disclosure features a method for processingmicrospheres. A suspension of solidified microspheres comprising polymerand active agent is circulated in a vessel. The suspension is moved fromthe vessel to a fluid inlet of a hydrocyclone, the hydrocyclone furtherincluding a first fluid outlet and a second fluid outlet. A flow of thesuspension having concentrated microspheres is removed from the secondfluid outlet. A flow of a relatively large amount of a liquid comparedto the flow from the second fluid outlet is removed from the first fluidoutlet.

Referring to specific aspects of the third embodiment, a pump isdisposed between the vessel and the hydrocylone. The pump is used topump the suspension from the vessel to the fluid inlet under pressure. Amixer can be in fluid communication with the vessel. The polymer,solvent and the active agent can be combined in the mixer to form anemulsion. The emulsion can be solidified in the vessel to form thesuspension in the vessel. Alternatively, another mixer can be in fluidcommunication with the vessel. The polymer, solvent and active agent canbe combined in the mixer to form the suspension and the suspension canbe directed from the mixer to the vessel. The emulsion or suspension canbe formed by mixing a dispersed phase comprising polymer, solvent andactive agent with a continuous phase. The continuous phase can beaqueous or not.

A fourth embodiment of this disclosure features a method forconcentrating microspheres comprised of polymer and active agentcomprising: feeding into a mixer a dispersed phase and a continuousphase, the dispersed phase including solvent, polymer and an activeagent; mixing the dispersed phase and the continuous phase in the mixerto make a suspension of microspheres; moving the suspension from themixer to a fluid inlet of a hydrocyclone, the hydrocyclone furtherincluding a first fluid outlet and a second fluid outlet; removing fromthe second fluid outlet a flow of the suspension having concentratedmicrospheres; and removing from the first fluid outlet a flow of arelatively large amount of a liquid compared to the flow from the secondfluid outlet.

Now, specific aspects of the fourth embodiment will be described. Themethod can comprise: moving the suspension from the mixer to a vesseland stirring the suspension in the vessel; adding water or a suspendingmedium to the vessel; and moving the suspension from the vessel to thefluid inlet.

The method can comprise removing from the flow from the first fluidoutlet a relatively large amount of the continuous phase, the water orthe suspending medium.

The method can comprise removing from the flow from the first fluidoutlet a relatively large amount of fine microspheres compared to theflow from the second fluid outlet.

The method can comprise providing a second hydrocyclone (HC-2) having afluid inlet, a first fluid outlet and a second fluid outlet, comprising:moving the suspension from the first fluid outlet of the hydrocyclone(HC-1) to the HC-2 fluid inlet; removing from the HC-2 second fluidoutlet a flow of the suspension having concentrated microspheres;removing from the HC-2 first fluid outlet a flow of a relatively largeamount of liquid compared to the flow from the HC-2 second fluid outlet;and combining the concentrated suspension from the HC-1 second fluidoutlet and from the HC-2 second fluid outlet to form a combinedsuspension.

The method can comprise removing from the flow from the HC-2 first fluidoutlet a relatively large amount of fine microspheres compared to theflow from the HC-2 second fluid outlet.

The method can comprise passing the microsphere suspension from thehydrocyclone to a second hydrocyclone in series or parallel with thehydrocyclone.

The method can comprise passing the flow of the microsphere suspensionfrom the second fluid outlet of the hydrocyclone to a hollow fiberfilter.

The method can comprise providing a solvent removal vessel (SRV); movingthe combined suspension into the SRV; and removing solvent from thecombined suspension in the SRV to form a solvent-removed suspension.

The method can comprise adding water or a suspending medium into thesolvent removal vessel (SRV).

The method can comprise: providing a hollow fiber filter (HFF) to removesolvent from the combined suspension in the SRV, the HFF including a HFFinlet, a first HFF outlet and a second HFF outlet; moving thesolvent-removed suspension from the solvent removal vessel (SRV) to theHFF inlet; removing water or the suspending medium from the first HFFoutlet to form a filtered suspension, and moving the filtered suspensionfrom the second HFF outlet to the SRV.

The method can comprise: providing a wet sieve including a suspensioninlet, a liquid outlet and a microsphere outlet; adding water or asuspending medium to the sieve. The suspension inlet of the sievereceives the concentrated suspension. The microspheres are removed fromthe microsphere outlet of the sieve and the liquid is removed from theliquid outlet.

Many additional features, advantages and a fuller understanding of theinvention will be had from the accompanying drawings and the detaileddescription that follows. It should be understood that the aboveTechnical Summary provides a description in broad terms while thefollowing Detailed Description provides a more narrow description andpresents embodiments that should not be construed as necessarylimitations of the broad invention as defined in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a perspective view of a hydrocyclone; FIG. 1B is a side viewof the hydrocyclone; FIG. 1C is a vertical cross-sectional view of thehydrocyclone of FIG. 1B and FIG. 1D is a top view of the hydrocyclone asseen along 1D-1D of FIG. 1B;

FIG. 2 is a flow diagram for an improved pharmaceutical productionprocess of sustained release microspheres with a hydrocyclone to removecontinuous phase (CP), wash microspheres, and formulate in a suspendingmedium to prepare a final dosage form;

FIG. 3 is a process flow diagram of an improved pharmaceuticalproduction process of sustained release microspheres with thehydrocyclone in use along with a hollow fiber filter to remove CP, washmicrospheres, and formulate in a suspending medium to prepare a finaldosage form;

FIG. 4 is a flow diagram of an improved pharmaceutical productionprocess with the hydrocyclone in use along with dead-end filtration toremove CP, washing microspheres, and drying to obtain sustained releasemicrospheres. Drying is typically conducted by blowing a stream of airor nitrogen.

FIG. 5A is a particle size distribution curve for a hydrocyclone 1(HC-1) underflow fraction and this is compared in FIG. 5B to theparticle size distribution curve for a hydrocyclone 3 (HC-3) overflowfraction while processing doxycycline microspheres using hydrocyclones;

FIG. 6 is a doxycycline level in rat blood while injecting doxycyclinemicrospheres processed by a hollow fiber filter (HFF) alone at the doseof 10 mg drug/Kg rat; and

FIG. 7 is an in-vitro release comparison of doxycycline microspheresprocessed by a hydrocyclone and hollow fiber filter (HC-HFF) versus ahollow fiber filter (HFF) alone performed under physiological conditions(PBS, 37° C.).

DETAILED DESCRIPTION

Referring to FIG. 1, a hydrocyclone 10 includes a body 12 having a topportion 14 including a fixed inlet 16, a fixed overflow outlet 18 and afixed underflow outlet 20. The hydrocyclone is tubular so that theinterior 22 of the hydrocyclone is hollow. The hydrocyclone has a centerspace 23 surrounded by interior walls 24 of the hydrocyclone. Thehydrocyclone is in the form of a cylinder having a straight section 26where the walls on the exterior are the same diameter throughout themiddle portion of the hydrocyclone. The top portion includes a straightsection portion or a vortex finder 28 extending from the interior to theoverflow outlet 18. The interior of the hydrocyclone also includes anangled bore 30 which slopes from the straight section at the middleportion 32 of the hydrocyclone to a decreasing diameter near a bottomportion 34 of the hydrocyclone. The interior surface of the angled boreleads to the outside of the hydrocyclone at the underflow outlet. Thevortex 36 receives a flow of material from the outside via inlet 16under pressure (i.e, under positive pressure such as 10 psi, wherein theactual pressure for operation would be optimized). The release of thematerial against the angled bore 30 separates the flow of material intotwo different pathways towards the top portion 14 and bottom portion 34of the hydrocyclone. Heavier or larger particles travel downward andoutward along the main vortex 36 in the interior and leave through theunderflow outlet 20. A small proportion of lighter or finer particlestravel upward along a center of the main vortex at 38 and leave theinterior of the hydrocyclone near its top portion through the overflowoutlet 18. A majority of the fluid in the hydrocyclone leaves throughthe overflow outlet. This fluid may include water, continuous phase ordiluents or a combination thereof.

FIG. 2 shows a process flow diagram of the improved method forprocessing a microsphere suspension using hydrocyclones. The microspheresuspension is processed by removing the continuous phase (CP) along withsolvents and non-encapsulated drug, washing the microspheres with waterand also suspending the washed microspheres with an appropriateformulation solution. In the flow chart, 50 is a continuous phase (CP)source and 52 is a dispersed phase (DP) source. A mixer 54 produces amicrosphere suspension 56 from the DP and CP. Tubing 58, 60 permits flowof the CP and DP from the sources of CP and DP into the mixer. It shouldbe appreciated that the tubing entry locations into the mixer andlocations relative to the other devices of the systems of thisdisclosure are only schematic and can be different in actual practice.The microsphere suspension 56 from the mixer enters a solvent removalvessel 62 (SRV). Tubing 64 permits travel of the microsphere suspension56 from the mixer 54 to the solvent removal vessel 62. For the deliveryof CP and DP appropriate delivery pumps or devices are used (not shown).

The suspension in the vessel is delivered by a pump 68 to thehydrocyclone 69. The pump 68 is located along tubing 70 from the solventremoval vessel 62 to the inlet 16 of the hydrocyclone 69. Depending uponthe extent of CP removal from the suspension and also the removal ofsmall particles from the system, more than one hydrocyclone could beused. The flow chart describes the use of two hydrocyclones in series.The underflow 72 from the underflow outlet 20 is a concentratedsuspension of microspheres and the overflow 74 from the overflow outlet18 is mainly continuous phase CP with a small amount of smallermicrospheres. Tubing 76 extends from the overflow outlet 18 of the firsthydrocyclone 69 to the inlet 16 of a second hydrocyclone 78. Theoverflow 74 is further delivered along tubing 76 into the inlet port 16of the second hydrocyclone 78 (“HC-II”). No additional pump is necessaryfor this delivery. If necessary, pressure gauges could be introduced atthe inlet ports to monitor the flow for process control and monitoringpurposes. From the second hydrocyclone 78 (HC-II) the concentratedsuspension will flow as underflow 80 from the underflow outlet 20 andoverflow 82 will leave through the overflow outlet 18 as a waste stream.Additional hydrocyclones could be used in series or in parallel forprocessing aids.

Tubing 84 extends from the underflow outlets 20 of the hydrocyclones 62and 78 into the solvent removal vessel 62. Under-flows 72 and 80 areconcentrated suspensions that are combined and travel along the tubing84 to reach back into the solvent removal vessel 62 (SRV). A pump 85extends along tubing 86 from a wash water reservoir 87 to the solventremoval vessel 62. Washing water from the wash water reservoir 87 can bepumped along the tubing 86 into the SRV 62 using pump 85. For someprocesses, water is also added simultaneously with the microspheresuspension during the microsphere formation step. The inlet from theSilverson mixer can be closed to enable the concentration of HC treatedmicrospheres to increase and continuous phase to decrease. Thisoperation is for the situation in which the hydrocyclone is used forremoving excess CP and smaller microspheres, the microspheres are washedwith water and then performing diluent exchange. At the end, thesuspension of microspheres in diluent in tank 62 is ready to fill intovials and freeze dry, or ready for bulk freeze drying and can leave viathe outlet 63.

There are certain flow rate requirements to perform the entire operationin a confined space so that the volume does not outgrow in the SRV 62.In general, the combined flow rate of the microsphere suspension alongtubing 64 and wash water along tubing 86 should be similar to flow rateof overflow through 82. Even though the flow rate mentioned above isdesirable, it is not necessary when the capacity of the SRV 62 issufficiently large and capable of accommodating the increasing volume.During the microsphere washing step there will not be any flow through64. At this point the flow rate through 86 should be equal to 82.

After washing the microspheres with water, appropriate diluent(suspending medium) can be introduced instead of water. 88 is a diluentreservoir and 90 is a diluent input pump. The diluent pump 90 extendsalong tubing 92 from the diluent reservoir 88 to the solvent removalvessel 62. The diluent is pumped using pump 90 from the diluentreservoir 88 along the tubing 92 to the SRV 62. Performing thisoperation will suspend the microspheres in an appropriate suspendingmedium. Again, flow rate through 92 should be matched to 82 to maintainconstant volume in the SRV. The concentration of the microspheres can beincreased by continuing the hydrocyclone operation without input intothe SRV through the stream along tubing 64, or decreased by notperforming the hydrocyclone treatment (i.e., not pumping using pump 68)while adding diluents through the stream along tubing 92. This processwill allow the achievement of final formulation with appropriate potencyof the drug. The suspension can be further filled into vials and freezedried. The suspension can also be freeze dried directly to achieve theproduct as a bulk powder. Filling is carried out asceptically by astandard procedure adopted by the industry. The filling machine isautomated; vials will be open to laminar flow of HEPA filtered air andthe procedure is qualified by media fill.

The diluent components of this disclosure may include a viscosityenhancer, a small molecule solute as a lyoprotecting agent, and asurfactant. The viscosity enhancer is a water soluble polymer that helpsto suspend the particles and slows the particle settling if not mixed.

Suitable viscosity enhancing polymers are carboxy methyl cellulose(CMC), hydroxy propyl cellulose (HPC), hydroxy propyl methyl cellulose(HPMC), sodium carboxy methyl cellulose (SCMC), ethyl cellulose, hydroxymethyl cellulose, cellulose acetate phthalate, hydroxy methyl celluloseacetate succinate, maleic anhydride copolymer, hyalauronic acid,gelatin, high molecular weight polyethylene glycol, and combinationsthereof. In one embodiment, carboxy methyl cellulose (CMC) may be usedas a viscosity booster as part of the diluent formulation.

The lyoprotecting agent is a small molecule solute, for example,mannitol, sorbitol, sucrose, lactose, galactose, maltose, trehalose,dextrose, fructose, other sugar derivatives, cyclodextrins, and mixturesthereof. Polysaccharides such as starch, dextran, agar, xanthan, andmixtures thereof may be used. Stabilizers such as polyvinyl alcohol,polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyglutamicacid, and mixtures thereof may be used. The cryoprotecting agent mayalso be used to adjust the tonicity of the solution. In a firstembodiment, mannitol is used as the lyoprotecting agent. Thelyoprotecting agent may also be helpful to protect the isotonicity ofthe suspension for injectability.

The surfactant is a wetting agent that is selected from a group ofbiocompatible surfactants. Polysorbate 80 can be used in one embodiment.Other suitable surfactants include sodium cholate, sodium taurocholate,sodium taurodesoxycholate, lecithin, phospholipids, poloxamers, sorbitanesters, esters of fatty acids with polyoxyalkylene glycols,glycerol-polyalkylene stearate, homo- and copolymers of polyalkyleneglycols, Pluronic F68, Tween 20, Tween 40, Tween 80, Span 20, Span 40,Span 60, Span 80, HCO-50, HCO-60 emulsifiers such as gelatin, lowmolecular weight alcohols such as propylene glycol, and low molecularweight fatty acids.

Diluent could be sterile filtered to use in the formulation. Thecomposition is processed as a suspension and is substantially free fromaggregation. The suspended microspheres are easily freeze dried andcompatible with agents added. The composition is also found to havereduced water content providing greater stability for long term storage.

FIG. 3 shows a process flow diagram in which a microsphere suspension isreceived in an intermediate vessel (IMV), processed by a hydrocyclonefor concentrating microspheres by removing the continuous phase (CP) andsubsequently processing the suspension by a hollow fiber filter (HFF)into a solvent removal vessel (SRV). In FIG. 3, 100 is a source of thecontinuous phase (CP) and 102 is a source of the dispersed phase (DP). Amixer 104 produces a microsphere suspension 106 by mixing the DP and theCP. Suitable mixers are disclosed in U.S. Pat. No. 5,945,126, which isincorporated herein by reference. Tubing 108 extends from the source ofthe continuous phase 100 into the mixer 104 and tubing 110 extends fromthe source of the dispersed phase 102 into the mixer. Tubing 112 extendsfrom the mixer to an intermediate vessel (IMV) 114. The microspheresuspension from the mixer travels along the tubing 112 and enters theintermediate vessel (IMV) 114. It may be possible to pass the suspensioncoming out of the Silverson mixer directly to the hydrocyclone only ifthe suspension coming out of the mixer has sufficient positive pressure.Instead, it is easier to receive in an intermediate vessel and controlthe pressure using pump 116. This will provide good flexibility. For thedelivery of CP and DP appropriate delivery pumps or devices may be used(not shown).

The microsphere suspension in the intermediate vessel (IMV) is deliveredby a pump 116 into the inlet of a first hydrocyclone (HC-1) 120. Thepump 116 is located along tubing 118 that extends from the intermediatevessel to the inlet 16 of the first hydrocyclone 120. Depending upon theextent of CP removal from the suspension and the removal of smallparticles from the system, more than one hydrocyclone can be used. Theflow chart describes the use of two hydrocyclones in series. However,the process is not limited to two hydrocyclones. Underflow 122 from theunderflow outlet 20 of the first hydrocyclone 120 is a concentratedsuspension of microspheres and overflow 124 from the overflow outlet 18of the first hydrocyclone is mainly a relatively large volume of CP witha small amount of smaller microspheres. Tubing 126 extends from theoverflow outlet 18 of the first hydrocyclone 120 to the inlet 16 of asecond hydrocyclone 128. The overflow 124 is further delivered along thetubing 126 into the inlet 16 of the second hydrocyclone 128 (HC-2). Noadditional pump is necessary for this delivery. If necessary, pressuregauges could be introduced at the inlets to monitor the flow for processcontrol and monitoring purposes. From the second hydrocyclone (HC-2)128, the concentrated suspension will flow as underflow 130 from theunderflow outlet 20 and overflow 132 leaves the overflow outlet 18 as awaste stream.

Tubing 134 extends from the underflow outlets 20 of the first and secondhydrocyclones 120, 128 to a solvent removal vessel (SRV) 136. Theunderflow 122, 130 from the hydrocyclones HC-1 120 and HC-2 128(concentrated suspension) is combined and travels to the SRV 136 alongtubing 134. From the SRV, the suspension is re-circulated using a pump138 through a hollow fiber filter (HFF) 140. The hollow fiber filterincludes a body 142 having an inlet port 144, a permeate removal port146 and an outlet port 148. The pump 138 is disposed along tubing 150that extends from the solvent removal vessel SRV to the inlet 144 of thehollow fiber filter HFF. While the suspension is re-circulating throughthe HFF loop, permeate is removed through the port 146. A pump 152 canbe used to remove the permeate. The removal rate of permeate can bematched to the under flow from the hydrocyclones to maintain the volumelevel in the SRV. It is not necessary to match the flow, if the SRVvolume allows. Tubing 153 extends from the outlet 148 of the hollowfiber filter to the SRV 136. The microsphere suspension having permeate(e.g., continuous phase, water or diluent) removed travels from thehollow fiber filter along the tubing 153 into the SRV.

Tubing 156 extends from a wash water source 154 to the intermediatevessel 114 and tubing 158 extends from the wash water source 154 to thesolvent removal vessel 136. A pump 159 extends along the tubing 156,158. After the microsphere formation step is completed, the microspheresin the SRV 136 can be washed with water that travels from the watersource 154 along the tubing 158 into the SRV. Water can be added to themicrosphere suspension in the IMV 114 also if the process requiresimmediate water dilution during microsphere formation. The water wouldtravel from the water source 154 along the tubing 156 and to theintermediate vessel 114. A pump 162 is located along tubing 164 thatextends from the diluent storage vessel 160 to the solvent removalvessel 136. Microspheres can be recovered from the SRV 136 via outlet137 as a bulk and dried or they can be suspended in appropriateformulating agent (diluent) pumped from diluent storage vessel 160 alongtubing 164. Suspending the microspheres in diluents could be performedin the SRV itself or in another vessel using another HFF. Themicrosphere suspension in water or in diluents removed from the SRV viaoutlet 137 could be filled in vials and freeze dried by the standardtechniques.

FIG. 4 is a similar process to FIG. 3, except it uses a wet sieveapparatus 180 instead of hollow fiber filter (HFF) to process themicrospheres and does not include the SRV. Similar parts have similarreference numerals throughout the several views of this disclosure.Tubing 134 from the underflow outlets 20 of the first and secondhydrocyclones 120, 128 leads to the wet sieve 180. The underflow streams122, 130 from the first and second hydrocyclones 120, 128 are combinedand travel along the tubing 134 to the wet sieve. Wash water from source154 can be pumped by pump 159 into the sieve unit and water will bedrained through port 182. Microspheres can be dispensed as a slurrythrough 184. Also, the microspheres in the sieve unit could be dried inthe sieve itself by flowing dry air or inert gas at appropriatetemperature and the dried microsphere could be collected as a bulkpowder through port 184. SWECO sieve units operate under a parameter(unique vibration pattern) that allows the dry particles (or slurry) todischarge efficiently.

Evaluation of the Hydrocyclone for Formation of Sustained ReleaseMicrospheres

The efficiency of hydrocyclone operation can be determined by theconcentration coefficient and yield of microspheres through theunderflow. Concentration coefficient is the ratio of particleconcentration in the underflow to the feed suspension. A higherconcentration coefficient means effective separation of particles fromthe continuous phase. A higher inlet flow of suspension will result in ahigher inlet pressure in the hydrocyclone. A higher inlet pressureprovides a higher concentration coefficient. For microspheremanufacturing a higher concentration coefficient of particles isrelevant. A flow rate ratio between underflow and overflow is a factorthat should be considered along with efficient separation. If theunderflow is low it is beneficial for the process since relatively largeamounts of water can be removed from the system quickly through theoverflow. However, there is a possibility of losing a fraction ofparticles in the overflow. Appropriate hydrocyclone type, and itsmanufactured apex and inlet flow should be selected for good yield ofparticles along with efficient removal of water from the system. Yieldof microspheres through the underflow should also be considered,especially while processing expensive pharmaceutical formulations. Yieldis the ratio between the amount of microspheres obtained through theunderflow outlet compared to the amount through the inlet. The amount ofmicrospheres is a combination of concentration of microspheres and flowrate of the suspension. If necessary, multiple hydrocyclones in seriescan be used to improve yield and to achieve a desired yield.

Removal of smaller particles occurs through the overflow. The range ofparticle size of small particles to be removed is based on the type ofhydrocyclone, and its manufactured apex and flow rate.

Example 1 Processing Microsphere Suspension Using a Hydrocyclone: Effectof Inlet Flow Rate, Apex ID and Particle Concentration on ConcentrationCoefficient and Particle Size

A microsphere suspension was prepared from a polylactide-co-glycolidesolution in dichloromethane by an oil-in-water process using an in-lineSilverson mixer as per U.S. Pat. No. 5,945,126. Polyvinyl alcoholsolution (0.35%) was used as the continuous phase. The Silverson mixingspeed was 7000 rpm. Microparticles were washed to remove residualsolvents and the particles were finally suspended in water. Theconcentration of microspheres in the suspension was 49 mg/g.Subsequently, suspensions having lower concentrations as shown in Table1 were also prepared by diluting it further in water. The suspension wasmaintained in a 20 L Applikon vessel (intermediate vessel, IMV) duringthe study and continuously stirred to maintain homogeneity. Suspensionsamples from the IMV were taken for concentration and particle sizeanalysis before hydrocyclone processing.

The suspension was pumped using a peristaltic pump at the flow ratesshown in Table 1 into the inlet port of the hydrocyclone. HydrocycloneType GMAX1U-3125 having Apex 118 received from Krebs Engineers was used.The suspension from a 20 L Applikon (IMV) was pumped to the hydrocycloneinlet and samples were obtained from two outlets; the bottom outletwhich produced the underflow retainent stream and top outlet which isthe overflow. In these experiments, underflow was approximately 20% ofthe overflow, which varied slightly, however, based on the conditions.

Table 1 shows the results. As the flow rate increases the concentrationof the microspheres in the retainant (underflow) increased. For thesuspension having the particle concentration of 49 mg/g at the inletflow rate of 10 L/min the concentration of the microspheres in theunderflow stream was 2.5 times compared to the bulk suspension. Thus, byprocessing a 20 L suspension through the hydrocyclone approximately 50%of the particles were collected in approximately 2 L (20% of the volume)in 2 minutes duration. A higher inlet flow rate produced the underflowstream at even higher concentrations. Experiments using dilutesuspension showed even higher concentration coefficients as shown inTable 1. Additionally, the particle size distribution (volumedistribution) showed that the underflow stream contained less smallerparticles since smaller particles were removed through the overflowstream. Particle size at 10% volume distribution showed up to 100%increase. Thus, particle size showed a considerable reduction of smallerparticles from the system.

TABLE 1 Effect of Particle Concentration and Flow Rate on ConcentrationCoefficient and Particle Size 10% Cumulative 50% Cumulative Conc. ratioin Volume Fraction Volume Fraction Conc. of Inlet Conc. of underflow(10% CVF) (50% CVF) Microspheres flow Inlet particles in versus in %Change % Change in IMV rate pressure underflow IMV (inlet Microncompared to Micron compared to (g/g) (L/min) (PSI) (g/g) flow) Size bulk(IMV) Size bulk (IMV) 0.049 Bulk sample from IMV 3.96 N/A 26.9 N/A 10.010 0.124 2.5 5.87 +48 30.0 +12 12.8 14 0.154 3.1 6.57 +66 30.0 +12 14.316 0.169 3.5 6.44 +63 29.7 +10 0.025 Bulk Sample from IMV 3.07 N/A 24.2N/A 10.0 8 0.074 3.0 5.82 +90 28.6 +18 12.8 13 0.089 3.6 6.16 +101 28.4+17 14.3 16 0.095 3.8 6.31 +106 27.7 +14 0.006 Bulk Sample from IMV 3.22N/A 24.9 N/A 10.0 7 0.017 2.8 5.85 +82 28.3 +14 12.8 12 0.020 3.3 6.45+100 28.5 +15 14.3 14 0.022 3.7 6.24 +94 27.8 +11

Example 2

The effect of the apex ID of the hydrocyclone on concentrationcoefficient and particle size of microspheres was evaluated usinghydrocyclone model GMAX1U-3125 (from Krebs) using three different apexIDs A (0.118), B (0.157), and C (0.197) from Krebs Studies wereperformed using the microsphere suspension having 15 mg/g particleconcentration at two different flow rates, 8.7 L/min and 12.6 L/min.When the hydrocyclone apex was changed from A to C, the concentrationcoefficient increased from 4.0 to 5.0 at 8.7 L/min flow rate and from4.9 to 6.7 at 12.6 L/min flow rate. Additionally, apex C removed asubstantial amount of small particles. In the bulk suspension (feed) 10%of the particles were under 2.76 micron. Upon processing through thehydrocyclone the underflow showed 10% of the particles were under 13microns in particle size. By selecting appropriate apex and inlet flowrate smaller particles could be eliminated to various extents from theproduct.

TABLE 2 Effect of Apex type on Concentration Coefficient and ParticleSize 10% particles 50% Conc. Conc. under particles under of ratio in % %particles under- Change Change in flow com- com- Inlet under- versuspared pared Flow flow in Micron to bulk Micron to bulk Apex (L/min)(g/g) IMV Size (IMV) Size (IMV) Bulk Sample from IMV 2.76 N/A 27.1 N/A(Conc. 0.015 g/g) A 8.7 0.060 4.0 7.41 168 38.2 41 12.6 0.074 4.9 7.83184 36.3 34 B 8.7 0.060 4.0 7.95 188 39.2 45 12.6 0.080 5.3 9.17 23238.1 41 C 8.7 0.075 5.0 10.6 284 41.1 52 12.6 0.100 6.7 13.0 371 40.5 49

Example 3 Doxycycline Microspheres Processed by Hollow Fiber Filter andHydrocyclone

Doxycycline is an antibiotic that requires sustained drug level in theblood for efficacy. Doxycycline microspheres for two week release wereprepared by processing the microspheres using a hollow fiber filter(HFF) process. Using the HFF process, the suspension produced by thein-line Silverson was approximately 50 L and took approximately 50minutes to eliminate 30 L water. Using the hydrocyclone it took only 4minutes to eliminate 30 L water. Also, the microspheres processedthrough the hollow fiber filter contained smaller particles. For thisproduct, the drug release kinetics performed by in-vitro and in-vivo(rat model) showed an initial burst. Microspheres processed through thehydrocyclone were found to be substantially free from small particles,which eliminated low drug containing small particles. The resultantmicrospheres showed considerably lower initial burst and higher drugload. The following procedure provides details.

Microsphere Preparation: Doxycycline containingpoly(d,l-lactide-co-glycolide) (PLGA) microspheres were prepared usingdoxycycline hyclate from Hovione and PLGA (RG503H) from BoehringerIngelheim by an oil-in-water process. The dispersed phase (organicphase) was prepared by dissolving 20 g doxycycline hyclate, 80 g RG503H(PLGA), 240 g methylene chloride and 60 g methanol. The solution wasthen filtered using a 0.2 micron PTFE (Satafluor 150 from Sartorious)filter. The continuous phase (aqueous phase) was 0.5% polyvinyl alcohol(PVA) in water prepared by dissolving 300 g low molecular weight PVA inhot water (70 to 90° C.). The solution was then cooled to ambienttemperature and water was added to reach 60 L in total volume. Thecontinuous phase was also filtered through 0.2 micron PVDF (Opticap XL10from Millipore). Doxycycline microspheres were prepared using an in-linemixer as described in U.S. Pat. No. 5,945,126 at a CP flow rate of 4L/min and a DP flow rate of 30 mL/min. The Silverson stirring speed was5000 rpm. A microsphere suspension produced at a rate of approximately 4L/min was collected in a 100 L tank and the suspension in theintermediate vessel (IMV) was stirred for suspension homogeneity.

Concentrating Suspension by Hollow Fiber Filter: SRV (3 L bioreactorfrom Applikon) equipped with inlet tubes for suspension, water anddiluent and also a re-circulation line through a Type 5A hollow fiberfilter (HFF from GE Healthcare) was used for processing the suspension.The suspension was pumped from an intermediate vessel IMV at 600 mL/min.The suspension in the SRV was re-circulated through the HFF at 6 L/minand permeate was removed at 600 mL/min. Thus, to remove 30 L water, ittook approximately 50 minutes. To process the entire suspension in theIMV, it took approximately 80 minutes. A suspension volume of 1.5 L wasmaintained in the SRV and the entire suspension was condensed into the1.5 L volume.

Concentrating Suspension by Hydrocyclone: Three hydrocyclones, TypeGMAX1U-3125 having apex IDs 0.197 (HC-1), 0.197 (HC-2), and 0.118 (HC-3)were connected in series. The outlet from the IMV was connected to theinlet of HC-1. Underflow from HC-1 was collected in a pre-weighed glasscontainer. Overflow from HC-1 was connected to the inlet of HC-2.Underflow from HC-2 was also collected in a pre-weighed glass container.Overflow from HC-2 was connected to the inlet of the HC-3. Underflowfrom HC-3 was collected in a pre-weighed glass container. Overflow fromHC-3 was a waste stream; however it was collected for particle size andparticle concentration measurement. Samples from individual containersincluding IMV were removed for analytical purposes. A suspension fromthe IMV was pumped at 13 L/min to the inlet of HC-1 using a peristalticpump (Watson Marlow). Under flow and overflow were collected. The totalvolume of underflow from HC-1, HC-2 and HC-3 was approximately 19 L.This was further transferred to the solvent removal vessel (SRV) using aperistaltic pump, processed using a hollow fiber filter eliminating CPat 600 mL/min condensing the microspheres in 1.5 L volume.

Washing of Microspheres: After concentrating the microspheres from 50 Lvolume to 1.5 L volume using the HFF or Hydrocyclone-HFF combination,the microspheres were washed as a suspension with room temperature waterfor 10 minutes, 35° C. water for 40 minutes (20 minutes at temperatureand a 20 minute ramp time) and again with room temperature water forapproximately 30 minutes. This was performed by volume exchanges asdescribed in U.S. Pat. No. 6,270,802.

Recovery and Freeze Drying: Washed microspheres were recovered on a PVDFmembrane using dead end filtration and freeze dried. Freeze drying wasperformed by freezing it to −35° C. over 1 hours and holding it at −35°C. for 3 hours, increasing the temperature from −35 to −5° C. over 2hours (≦150 mT vacuum), −5 to +5° C. over 6 hours (≦150 mT vacuum) and+5 to +30° C. over 2 hours (≦150 mT vacuum) and holding it at +30° C.for 8 hours (≦150 mT vacuum).

Microspheres prepared by the HFF process and by the hydrocyclone processwere characterized for drug content, particle size distribution and drugrelease. Microspheres prepared by the HFF process were evaluated forin-vivo drug release in rats. For the hydrocyclone process, drug loadand particle size were measured for individual fractions also forinformation purposes. Additionally, particle concentrations in varioussuspensions were also determined to evaluate the hydrocyclone efficiencycomparison.

Table 3 shows the performance of the hydrocyclone process. As shown, theHC processed the suspension at approximately 13 L/min while the HFFprocessed the suspension at 600 mL/min only. The hydrocyclone enablesprocessing relatively large volumes in a shorter duration compared tothe HFF due to the reasons previously described. A microspheresuspension could be processed at the same rate of production using a HCand a large holding vessel is not required. Hence, smaller processequipments could be used. For example, if the microsphere suspensionproduction rate is 15 L/min, the HC could easily remove CP at 15 L/min.The equipment is very compact (approx. 1 ft long 1 inch dia). However,to eliminate the CP at 15 L/min through HFF, there is no HFF availablethat operates at this rate, therefore 2 or 3 HFF would be placed inparallel and the re-circulation rate of the suspension through the HFFhas to be 150 L/min. This requires a large pump. Therefore, theequipment is large and cumbersome.

TABLE 3 Flow Rate, Concentration of Microspheres and Particle SizeDistribution HC-1 HC-2 HC-3 Flow rate Inlet flow 13.0 11.79 10.99(L/min) Underflow 1.81 0.80 0.84 Overflow 11.79 10.99 10.15 ParticleInlet 0.00185 0.00065* 0.00048* concentration Underflow 0.00906 0.001750.00119 (g/g) Overflow 0.00065* 0.00048* 0.0004* Particle Size for 10%Under 4.98 Inlet flow 25% Under 12.0 50% Under 30.2 75% Under 46.1 90%Under 59.7 Particle size for 10% Under 10.7 4.45 4.20 Underflow 25%Under 30.3 11.7 9.03 50% Under 44.6 28.8 24.6 75% Under 57.3 42.3 37.790% Under 68.3 53.8 48.4 Particle size for 10% Under 3.39 overflow 25%Under 5.94 50% Under 11.3 75% Under 23.6 90% Under 32.9 *Calculated

The results show that the concentration of doxycycline microspheres inthe underflow of the first HC was approximately 5 times that of theinlet and 14 times that of the overflow. Out of 96 g microspheresprocessed from the 50 L suspension, 66 g of microspheres were collectedin a 7.2 L suspension in the HC-1 underflow. Thus, 69% of thedoxycycline microspheres were collected in 17% of the volume and theseparticles are substantially free from smaller particles. This wasachieved rapidly within 4 minutes of processing.

The concentration of microspheres in the underflow of the second HC was2.7 times the particle concentration of the inlet and 3.7 times comparedto the overflow. The results show that 8.2 g (9% of bulk) were recoveredthough the HC-2 underflow in 4.7 L volume. Thus 9% of microspheres inthe original bulk (in IMV) were collected in 9% volume. Concentration ofthe microspheres in the underflow of HC-2 is similar to that of theoriginal suspension in the IMV. Even though there is no net gain onparticle concentration, this fraction could be collected for good yield,if needed.

Concentration of microspheres in the underflow of the HC-3 was 2.4 timesthe concentration of the particles in the overflow. Concentration of theparticles in the underflow is even lower, lower than that of the bulksuspension in the IMV. Again, this fraction could be included in theproduct, if yield of the particles is critical. The concentration ofparticles in the underflow decreases with added HC in series because theconcentration of particles entering into the subsequent HC is lower andlower compared to the concentration in the IMV. Multiple HC areintroduced here for the purpose of good yield only.

Particle size also showed a large difference among the portions receivedat the HC underflow and overflow. Microspheres received through HC-1underflow had a small amount of small particles (FIG. 5A) and the HC-3overflow contained a majority of the smaller particles (FIG. 5B).

For the doxycycline microsphere run, underflow from all three HCs weredelivered to the SRV and further processed (concentrated and washed)using the HFF. Resulting microspheres were compared with themicrospheres prepared by HFF filter alone for drug load, particle sizeand drug release properties. Since HC-2 and HC-3 underflow were alsocombined with the product there were some smaller particles in the HCprocessed microspheres; however, the amount of smaller particles is muchless compared to the HFF alone processed microsphere batch. Drug load inthe microspheres was 12.3% for the HC-HFF processed microspheres.HFF-alone processed microspheres showed only a 9.4% drug load. This wasprimarily due to the presence of smaller particles having low drug load.It was found that the particles in the overflow of HC-3 had only 4.3%drug load. Lower drug load microspheres are not preferred since itrequires more microspheres to be injected to provide a similar dose.

An additional benefit found in the HC-HFF processed microspheres was thereduction of initial burst release. HFF-alone processed microsphereswere injected in rats to follow the in-vivo release rate. Six rats wereinjected with the HFF processed microspheres at the dose of 10 mgdoxycycline/Kg rat. Five rats were used for the test group. Bloodsamples were taken at 2 hours, 6 hours, 1 day, 3 days, 6 days, 10 days,15 days and 21 days and the concentration of doxycycline in the bloodwas assayed by HPLC-MS. Results showed that HFF-alone processedmicrospheres had a huge initial burst (FIG. 6). These microspheres wereprepared to have approximately 10 days release duration. Low to moderateburst is desirable from these formulations. The higher burst shown inFIG. 6 is not desirable.

HFF-alone processed microspheres and HC-HFF processed microspheres weretested for in-vitro release under physiological conditions. This wasperformed with 20 mg microspheres in 20 mL phosphate buffered salinecontained in a 40 mL screw capped tubes placed in a 37° C. shaking waterbath. The entire supernatant was removed for assay at each samplingpoints, 1 hour, 5 hours, 1 day, 3 days and 7 days and replaced withfresh release media. Drug concentration in the supernatant was assayedfor doxycycline by UV spectrophotometer against calibration standards.Percentage of drug released at each time point was calculated from thetotal drug introduced into the sample tube compared to that found in therelease media. The results are shown in FIG. 7 as a plot for cumulativerelease against time. It was found that HC-HFF processed microsphereshave much lower initial burst (one-fifth) compared to the HFF aloneprocessed microspheres. Thus, by eliminating smaller particles that hadlow drug load and faster release, unwanted initial burst release wasreduced from the microspheres. The HC-HFF processed microspheres showedsteady state nearly zero-order release while the HFF alone processedmicrospheres showed five times higher initial burst and higher initialrelease during the initial five days.

Many modifications and variations of the invention will be apparent tothose of ordinary skill in the art in light of the foregoing disclosure.Therefore, it is to be understood that, within the scope of the appendedclaims, the invention can be practiced otherwise than has beenspecifically shown and described.

1. A system for processing microspheres comprising a vessel thatcontains a suspension of solidified microspheres comprising polymer andactive agent; and a hydrocyclone having a fluid inlet, a first fluidoutlet and a second fluid outlet, wherein said fluid inlet is in fluidcommunication with said vessel and receives said suspension, said secondfluid outlet contains a flow of said suspension having concentrated saidmicrospheres and said first fluid outlet contains a flow of a relativelylarge amount of liquid compared to said flow from said second fluidoutlet.
 2. The system of claim 1 comprising a pump disposed between saidvessel and said hydrocylone for pumping said suspension from said vesselto said fluid inlet under pressure.
 3. The system of claim 1 comprisinga mixer that combines said polymer, solvent and said active agent toform an emulsion, wherein said mixer is in fluid communication with saidvessel and said emulsion is solidified in said vessel to form saidsuspension in said vessel.
 4. The system of claim 1 comprising a mixerthat combines said polymer, solvent and said active agent to form saidsuspension, wherein said mixer is in fluid communication with saidvessel.
 5. A system for concentrating microspheres comprised of polymerand active agent, comprising a mixer into which a dispersed phase and acontinuous phase are fed, said dispersed phase including solvent,polymer and an active agent, said mixer including a mixing element whichmixes said dispersed phase and said continuous phase to make asuspension of microspheres; and a hydrocyclone having a fluid inlet, afirst fluid outlet and a second fluid outlet, wherein said fluid inletis in fluid communication with said mixer and receives said suspension,said second fluid outlet contains a flow of said suspension havingconcentrated said microspheres and said first fluid outlet contains aflow of a relatively large amount of liquid compared to said flow fromsaid second fluid outlet.
 6. The system of claim 5 comprising: a vesselfor containing and stirring said suspension; first tubing leading fromsaid mixer to said vessel; a source of water or a suspending medium;second tubing leading from said source to said vessel; a pump for movingsaid water or said suspending medium from said source along said secondtubing into said vessel; third tubing leading from said vessel to saidfluid inlet; and a pump for pumping said suspension from said vessel tosaid fluid inlet.
 7. The system of claim 6 wherein said flow from saidfirst fluid outlet contains a relatively large amount of said continuousphase, said water or said suspending medium as said liquid compared tosaid flow from said second fluid outlet.
 8. The system of claim 7wherein said flow from said first fluid outlet contains a relativelylarge amount of fine said microspheres compared to flow from said secondfluid outlet.
 9. The system of claim 6 comprising a second hydrocyclone(HC-2) having a fluid inlet, a first fluid outlet and a second fluidoutlet, wherein said HC-2 second fluid outlet contains a flow of saidsuspension having concentrated said microspheres and said HC-2 firstfluid outlet contains a flow of a relatively large amount of said liquidcompared to said flow from said HC-2 second fluid outlet, wherein saidfirst fluid outlet of said hydrocyclone (HC-1) is in fluid communicationwith said fluid inlet of said second hydrocyclone (HC-2) and saidconcentrated suspension from said HC-1 second fluid outlet and from saidHC-2 second fluid outlet are combined.
 10. The system of claim 9 whereinsaid flow from first fluid outlet of said second hydrocyclone (HC-2)contains a relatively large amount of said continuous phase, said wateror said suspending medium compared to said flow from said HC-2 secondfluid outlet.
 11. The system of claim 5 comprising a second hydrocyclonein series with said hydrocyclone.
 12. The system of claim 5 comprising asecond hydrocyclone in parallel with said hydrocyclone.
 13. The systemof claim 9 further comprising a solvent removal vessel (SRV) thatreceives said combined concentrated suspension from said HC-1 secondfluid outlet and from said HC-2 second fluid outlet and forms washedmicrospheres substantially free from solvents and unwanted materials.14. The system of claim 13 comprising a hollow fiber filter (HFF) havinga HFF inlet, a first HFF outlet and a second HFF outlet, fourth tubingbetween said solvent removal vessel (SRV) and said HFF inlet, and a pumpfor moving said solvent-removed suspension from said SRV, along saidfourth tubing to said HFF inlet and fifth tubing extending from saidsecond HFF outlet to said SRV, wherein permeate is removed from saidfirst HFF outlet, and filtered said suspension travels from said secondHFF outlet along said fifth tubing to said SRV.
 15. The system of claim14 comprising a second source of water or suspending medium, sixthtubing leading from said second source to said solvent removal vessel(SRV) and a pump for pumping said water or said suspending medium fromsaid second source along said sixth tubing into said SRV.
 16. The systemof claim 5 comprising a hollow fiber filter that receives saidmicrosphere suspension traveling from said second fluid outlet.
 17. Thesystem of claim 5 comprising a wet sieve having a suspension inlet, aliquid outlet and a microsphere outlet, a source of water or suspendingmedium, tubing leading from said source to said sieve, and a pump formoving said water or said suspending medium from said source along saidtubing into said sieve, wherein said suspension inlet of said sievereceives said concentrated suspension from said second fluid outlet. 18.A method for processing microspheres comprising: circulating asuspension of solidified microspheres comprising polymer and activeagent in a vessel; moving said suspension from said vessel to a fluidinlet of a hydrocyclone, said hydrocyclone further including a firstfluid outlet and a second fluid outlet; removing from said second fluidoutlet a flow of said suspension having concentrated said microspheres;and removing from said first fluid outlet a flow of a relatively largeamount of a liquid compared to said flow from said second fluid outlet.19. The method of claim 18 comprising a pump disposed between saidvessel and said hydrocylone, comprising using said pump to pump saidsuspension from said vessel to said fluid inlet under pressure.
 20. Themethod of claim 18 comprising a mixer in fluid communication with saidvessel, combining said polymer, solvent and said active agent in saidmixer to form an emulsion, and solidifying said emulsion in said vesselto form said suspension in said vessel.
 21. The method of claim 18comprising a mixer in fluid communication with said vessel, combiningsaid polymer, solvent and said active agent in said mixer to form saidsuspension and directing said suspension from said mixer to said vessel.22. A method for concentrating microspheres comprised of polymer andactive agent comprising feeding into a mixer a dispersed phase and acontinuous phase, said dispersed phase including solvent, polymer and anactive agent; mixing said dispersed phase and said continuous phase insaid mixer to make a suspension of microspheres; moving said suspensionfrom said mixer to a fluid inlet of a hydrocyclone, said hydrocyclonefurther including a first fluid outlet and a second fluid outlet;removing from said second fluid outlet a flow of said suspension havingconcentrated said microspheres; and removing from said first fluidoutlet a flow of a relatively large amount of a liquid compared to saidflow from said second fluid outlet.
 23. The method of claim 22comprising: moving said suspension from said mixer to a vessel andstirring said suspension in said vessel; adding water or a suspendingmedium to said vessel; and moving said suspension from said vessel tosaid fluid inlet.
 24. The method of claim 23 comprising removing fromsaid flow from said first fluid outlet a relatively large amount of saidcontinuous phase, said water or said suspending medium.
 25. The methodof claim 24 comprising removing from said flow from said first fluidoutlet a relatively large amount of fine said microspheres compared tosaid second fluid outlet.
 26. The method of claim 24 comprisingproviding a second hydrocyclone (HC-2) having a fluid inlet, a firstfluid outlet and a second fluid outlet, comprising moving saidsuspension from said first fluid outlet of said hydrocyclone (HC-1) tosaid HC-2 fluid inlet; removing from said HC-2 second fluid outlet aflow of said suspension having concentrated said microspheres; removingfrom said HC-2 first fluid outlet a flow of a relatively large amount ofliquid compared to said flow from said HC-2 second fluid outlet; andcombining said concentrated suspension from said HC-1 second fluidoutlet and from said HC-2 second fluid outlet to form a combinedsuspension.
 27. The method of claim 26 comprising removing from saidflow from said HC-2 first fluid outlet a relatively large amount of finesaid microspheres compared to said flow from said HC-2 second fluidoutlet.
 28. The method of claim 22 comprising passing said microspheresuspension from said hydrocyclone to a second hydrocyclone in serieswith said hydrocyclone.
 29. The method of claim 22 comprising passingsaid microsphere suspension from said hydrocyclone to a secondhydrocyclone in parallel with said hydrocyclone.
 30. The method of claim22 comprising passing said microsphere suspension from said second fluidoutlet of said hydrocyclone to a hollow fiber filter.
 31. The method ofclaim 26 further comprising providing a solvent removal vessel (SRV);moving said combined suspension into said SRV; and removing solvent fromsaid combined suspension in said SRV to form washed microspheressubstantially free from solvents and unwanted materials.
 32. The methodof claim 31 comprising adding water or a suspending medium into saidsolvent removal vessel (SRV).
 33. The method of claim 32 comprisingproviding a hollow fiber filter (HFF) to remove solvent from saidcombined suspension in said SRV, said HFF including a HFF inlet, a firstHFF outlet and a second HFF outlet; moving said solvent-removedsuspension from said solvent removal vessel (SRV) to said HFF inlet;removing water or said suspending medium from said first HFF outlet toform a filtered suspension, and moving said filtered suspension fromsaid second HFF outlet to said SRV.
 34. The method of claim 22comprising providing a wet sieve including a suspension inlet, a liquidoutlet and a microsphere outlet, adding water or a suspending medium tosaid sieve, wherein said suspension inlet of said sieve receives saidconcentrated suspension, removing said microspheres from saidmicrosphere outlet of said sieve and removing liquid from said liquidoutlet.